Spirochetes are a diverse group of helical and planar wave-shaped bacteria having a unique structure and mode of motility. Spirochetes are the causative agents of syphilis (Treponema pallidum) and Lyme disease (Borrelia burgdorferi) and are associated with periodontal disease (Treponema denticola). The periplasmic location of the flagellar filament, together with the cell shape, enables the spirochete to move through dense matrices that would inhibit most other bacteria and assists in pathogenesis. This proposal involves determining the function of motility-associated polypeptides, development and analysis of mutants altered in motility to determine gene regulatory mechanisms, and assessment of the virulence capabilities of motility mutants. Treponema denticola will be used as a model for treponemal motility because it possesses newly identified tools for genetic analysis. The first polypeptide encoded by the fla motility operon, Tap1, has no known homologs but the investigators hypothesize it is involved in motility. This hypothesis will be tested using targeted mutagenesis to inactivate tap1 to observe the effect on cell movement together with immune electron microscopy to determine cellular location. Analysis of transcription of a polar flgE mutant suggests that T. denticola also has a unique system for regulation of motility gene expression. To test this hypothesis, targeted non-polar mutations will be made in motility-associated genes of specific classes, including the flagellar switch (fliG), hook (flgE), and regulation (fliA). Cytoplasmic filaments are a major component of treponemal cells with unknown function. A T. denticola mutant that lacks cytoplasmic filaments was constructed by insertional inactivation and these cells possess altered motility in liquid media and reduced colony diameter on 0.5% agarose-NOS plates. They hypothesize that the treponemal cytoplasmic filaments play a role in motility either directly through interaction with the periplasmic flagellum or indirectly through maintenance of cell structure. Biochemical analysis and tomography, together with the analysis of the cfpA-interrupted mutants will ascertain the role of this major cellular polypeptide. Finally, the involvement of motility in the virulence capabilities of T. denticola will be assessed in a murine abscess model using the specific motility mutants. The broad long-term objective is to understand the structure, function and regulation of treponemal motility-associated polypeptides and to assess their role in pathogenesis. Understanding the relationship of motility and cell structure to spirochete pathogenesis will result in development of therapeutics targeted towards inhibition of spirochete motility for prevention of human disease.